Background/Rationale: Currently, there is no effective preventative vaccine for human immunodeficiency virus (HIV)-1, nor is there a cure for this chronic infection. At least one in 250 Veterans is infected with HIV. HIV-1 Clade B strains are the predominant strain in the US, and the overwhelming source of infection for Veterans (97% Clade B). The only treatment available is life-long therapy with combined anti-retroviral therapy. As part of our current Merit Award, we were successful in discovering a new family of broadly neutralizing antibodies (bNAbs) that are the most potent and broad described to date (N49 P series), including N49P7 (100% breadth in 117 virus panel, median IC50 0.10 ug/ml) and N4P9.3 (97% breadth in the 117 virus panel, median IC50 0.0495 ug/ml). Further, we identified HIV-1 envelope sequences from contemporaneous, circulating Clade B viruses from the same donor, thus delineating their resistance mutations. Other preliminary data include in silico data demonstrating antibody engineering, and in vivo data demonstrating the mouse models used. Objectives: Our hypothesis is that we can exploit both antibody and viral envelope sequences to engineer the N49 P series bNAbs towards even greater breadth and potency, thus covering variants with the potential to escape naturally occurring bNAbs of N49 and similar specificities. The specific aims of this proposal are 1) Engineer the N49 P series bNAbs to optimize clinical utility; and 2) Test the engineered bNAbs in two humanized mice models to establish in vivo efficacy against HIV-1. Methods: Our plan is to first develop a panel of engineered 60 mAbs from the N49 P series bNAbs. Engineering will be informed by crystallographic information regarding wild type N49P7 and N49P9.3 Fab- gp120 interactions; in silico modeling of N49P7 and N49P9.3 resistant Env variants found in standard screening panels; in silico modeling of N49P7 and N49P9.3 resistant Env variants found in the contemporaneous plasma that harbored the bNAb lineages. The aim will be to determine the key residues that are involved in neutralization (with a focus on Clade B strains), as well as those that are detrimental to stability. With this knowledge, we will create 60 mutants with the aim of improving potency/breadth, while maintaining or improving stability, lack of auto/polyreactivity, and ADCC. A list of 5 final bNAbs (in LS format) will be tested in humanized mice models for half-life, and in vivo efficacy (autologous and heterologous challenges in cell-free and cell-associated challenges in the Hu-PBL mouse model). Findings: Anticipated results will be isolation of new engineered bNAbs from the N49 P series family that will demonstrate improved potency and breadth, and adequate stability and PK. Furthermore, these bNAbs will be tested in a humanized mouse models for in vivo efficacy, further moving these products toward clinical use. Status: We have isolated the N49 P series of bNAbs. The team we have assembled has expertise in monoclonal antibody production, HIV-1 virus sequencing, biochemistry and antibody engineering, and mouse models of HIV-1 infection. Impact: If our hypothesis proves correct, these bNAbs will represent a clinical breakthrough and the lead candidate for antibodies being considered for HIV-1 prophylaxis and treatment, and could be used in a variety of clinical formats, especially at the VA, as HIV continues to be a problem for our Veterans.